Peptide inhibitors of protein synthesis

ABSTRACT

The present invention discloses compositions of peptide inhibitors of protein synthesis, and methods of identifying peptide inhibitors that are capable of inhibiting protein synthesis through an interaction at a stem-loop H18 in 16S rRNA of a 30S ribosomal subunit. Screening methods for peptides are disclosed, in addition to methods of determining the affinity of a test compound for a ribosomal subunit.

GOVERNMENT SUPPORT

The invention was supported by National Institutes of Health Grant No.:U19 AI56575. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention concerns the identification and use of novelpeptide inhibitors of protein synthesis. Specifically, screening methodsfor peptides that interfere with protein synthesis by binding to aribosomal subunit are disclosed.

BACKGROUND OF THE INVENTION

According to the U.S. Centers for Disease Control and Prevention over40,000 people in North America die each year from infections caused bydrug-resistant germs. Emerging bacterial resistance to currently knownclasses of antibiotics is a major worldwide health problem. In addition,the most commonly used antibiotics (e.g., macrolides, beta-lactams, andquinolones) were initially introduced more than thirty years ago.Antibiotic resistance is a critical health problem, aggravated by theemergence of multidrug-resistant bacteria, and requires urgent attentionby the scientific community.

The overuse of antibiotics have sped up evolutionary adaptations thatenable bacteria and other microbes, such as viruses, fungi, andparasites, to survive these drugs. In addition, recent studies haveshown that the common use of antibacterial products, such as soaps, handsantizers, and household, can lead to the development of tolerance forcertain antibiotics. For example, such “cross-resistance” has been shownbetween triclosan, a common chemical in antibacterial hand sanitizer,and drug resistance to isoniazid, an antibiotic used for treatingtuberculosis.

Drug resistance is an increasingly difficult problem in hospitals sincecritically ill patients are less able to fight off infections withoutthe help of antibiotics. Heavy use of antibiotics in these patientsselects for drug resistance strains of bacteria. Unfortunately, thisworsens the problem by producing bacteria with greater ability tosurvive in the presence of even the strongest antibiotics. These“superbugs” have even developed resistance to vancomycin, which was onceconsidered the “antibiotic of last resort”.

A key factor in the development of antibiotic resistance is the abilityof infectious organisms to adapt quickly to new environmentalconditions. Bacteria are single-celled organisms with a relatively smallnumbers of genes and can reproduce rapidly. Therefore, a mutation thathelps a microbe survive exposure to an antibiotic will quickly becomedominant throughout the microbial population. The increasing pace withwhich bacteria evolve drug resistance coupled with the lack of newclasses of antibiotics has driven the need for new research strategies.

Accordingly, there exists a need for new antibiotic targets, and methodsof identifying new antibiotics. In particular, new compositions capableof inhibiting protein synthesis and/or new antibacterials targeting newsites in the ribosome would satisfy a long-felt therapeutic need.

SUMMARY OF THE INVENTION

In one aspect, the invention provides methods for selecting a peptidehaving a desired affinity for a target nucleic acid sequence. The methodcomprises the steps of presenting a target nucleic acid sequence as anextended target molecule having an enzymatically cleavable moiety and anaffinity linker joined to the target sequence; exposing the taggedtarget molecule to a phage display library that provides a plurality ofpeptides such that a peptide/target molecule complex is formed if abinding peptide with an affinity for the target nucleic acid sequence ispresent in the library; isolating the peptide/target molecule complex bybinding the affinity linker component of the complex with a bindingpartner; and enzymatically cleaving the peptide and target nucleic acidsequence from the bound complex. The binding peptide can then beisolated or identified from the cleaved portion of the complex. In someembodiments, the method further comprises tagging the extended targetmolecule with an affinity linker. The method can further include washingat each step.

In some embodiments, the extended target molecule can be an RNA/organicmolecule construct, or an RNA/DNA construct. The target nucleic acidsequence can be a ribosomal subunit sequence. For example the targetnucleic acid sequence can be a mimetic of a stem-loop H18 in 16S rRNA ofa 30S ribosomal subunit. In some embodiments, the extended targetmolecule can be an RNA/DNA construct with substantially similar threedimensional structure to a stem-loop H18 in 16S rRNA of a 30S ribosomalsubunit. In a preferred embodiment, the enzymatically cleavable moietycan be DNA. The step of enzymatically cleaving the binding targetcompound can comprise using DNase I.

The combination of the affinity linker and the binding partner can beselected from the group consisting of biotin-avidin and/or streptavidin,lectin-saccharide, protein A and/or protein G-immunoglobulin constantregion, Tag peptide sequence-Tag antibody, an hapten-antibody, areceptor-ligand, and Ni-NTA. In some embodiments, the binding partner isimmobilized, such as, but not limited to, immobilized onto a solidsupport, such as a bead, a surface, column, a magnetic bead, amagnetized particle, or a microtiter plate. For example, the RNA/DNAhybrid-ligand can bind specifically to the phage on one site, and to asolid support (e.g., beads or wells) on the ligand site. The release ofthe captured phage-RNA/DNA complex from the solid support can be doneusing enzymatic digestion.

In some embodiments, the identified test compound can be anantimicrobial compound which interacts with a ribosomal subunit so as toinhibit protein synthesis.

In another aspect, the invention provides compositions comprising apeptide selected from Table 1 and Table 2 and analogs and derivativesthereof, wherein the peptide is capable of inhibiting protein synthesis.The composition can further include a pharmaceutically acceptablecarrier. The peptide is capable of binding to a stem-loop H18 in 16SrRNA. In some embodiments, the peptide is coupled to a moiety capable ofdirecting the peptide to a target cell. In other embodiments, thecomposition further comprises a combination of the identified peptidesand one or more drug. For example, in some embodiments, one or moreantimicrobial or antibacterial can be combined with the peptides. Inpreferred embodiments, the drugs have a synergistic effect.

In another aspect, the invention discloses an isolated therapeuticpeptide composition comprising a sequence of at least seven amino acidscapable of inhibiting protein synthesis through an interaction at astem-loop H18 in 16S rRNA of a 30S ribosomal subunit. In someembodiments, the peptide exhibits at least about 25 percent inhibitionof protein synthesis in a cell-free translational assay, or at leastabout 30 percent, or preferably at least about 50 percent, or preferablyat least about 60 percent, or preferably at least about 70 percent, ormore preferably at least about 80 percent inhibition of proteinsynthesis in a cell-free translational assay. The peptide is in therange of about seven to about thirty amino acid residues and selectivelybinds to the stem-loop H18 in 16S rRNA of the 30S ribosomal subunit. Insome embodiments, the peptide is in the range of about seven to abouttwenty amino acid residues, or in the range of about five to abouttwelve amino acid residues. The peptide sequence can include at leastone sequence selected from the group consisting of SEQ ID NOS: 2-33. Insome embodiments, the peptide sequence includes at least one of theamino acid motifs selected from the group consisting of AMS, HPP, THP,LHL, SXPXP (SEQ ID NO: 6) and SXXLPT (SEQ ID NO: 7). Preferred peptidesequence include, for example, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ IDNO: 28. In some embodiments, the therapeutic peptide composition furtherincludes a pharmaceutically acceptable carrier. In other embodiments,the peptide can be coupled to a moiety capable of directing the peptideto a target cell.

In some embodiments, the invention discloses isolated therapeuticpeptides consisting of a sequence of no more than 30 amino acidresidues, said sequence of amino acid residues including at least onesequence selected from Tables 1 and 2 (SEQ ID Nos: 2-33). In someembodiments, the therapeutic peptides have between about 7 amino acidresidues to about 30 amino acid residues, or preferably between about 7amino acid residues to about 20 amino acid residues, or more preferablybetween about 7 amino acid residues to about 12 amino acid residues. Inpreferred embodiments, the invention discloses isolated therapeuticpeptides consisting of a sequence of no more than 30 amino acidresidues, said sequence of amino acid residues including the three aminoacid motif AMS. In other embodiments, the invention discloses isolatedtherapeutic peptides consisting of a sequence of no more than 30 aminoacid residues, said sequence of amino acid residues including at leastone of the amino acid motifs selected from the group consisting of AMS,HPP, THP, LHL, SXPXP (SEQ ID NO: 6) and SXXLPT (SEQ ID NO: 7). In otherembodiments, the invention discloses isolated therapeutic peptidesconsisting of a sequence of no more than 30 amino acid residues, saidsequence of amino acid residues including at least one of SEQ ID NOS:2-33, preferably at least one of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ IDNO: 28.

In another aspect, the invention provides a method for treating abacterial infection in a subject comprising administrating a peptidecapable of inhibiting protein synthesis through an interaction at astem-loop H18 in 16S rRNA of a 30S ribosomal subunit. The peptide can,for example, be selected from Tables 1 and 2, or derivatives or isotopesthereof, or other peptides identified using the method of the presentinvention. The peptide can be delivered in an amount sufficient toinhibit the growth of bacteria in vivo. Non-limiting examples of abacteria include, for example, Gram-positive bacteria such as Bacillusanthracis, Actinomyces bovis, Enterococcus fetalis, Hemophiluspneumoniae, Listeria monocytogenes, Mycobacterium tuberculosis, M.leprae, M smegmatis, Proprionibacterium acnes, Sarcina ventriculi,Staphylococcus aureus, S. epidermis, S. intermedias, Streptococcushemolyticus, & pneumoniae; Gram-negative bacteria such as Campylobacterfetus, Erwinia carotovora, Flavobacterium meningosepticum, Helicobacterpylori, Hemophilus pneumoniae, H. influenzae, Klebsiella pneumonia,Neisseria gonorrhoeae, Pseudomonas aeruginosa, Shigella dysenteria,Salmonella typhi, S. paratyphi, Yersinia pestis, Escherichia coliserotype 0157, and Chlamydia species, Helicobacter species. In someembodiments, the peptide is delivered locally or regionally to a site ofinfection. The peptide can administered to a wound site, appliedtopically, delivered systemically, delivered via intravenous orintraarterial injection. The method can further include administering tothe subject one or more antimicrobial compounds.

In another aspect, the invention provides a method for preventing amicrobial infection in a subject comprising administrating a peptidecapable of inhibiting protein synthesis through an interaction at astem-loop H18 in 16S rRNA of a 30S ribosomal subunit in an amountsufficient to inhibit the growth of microbes in vivo.

In another aspect, the invention method for preventing microbial growthin a solution comprising mixing said solution with a peptide capable ofinhibiting protein synthesis through an interaction at a stem-loop H18in 16S rRNA of a 30S ribosomal subunit in an amount sufficient toinhibit the microbial growth in said solution.

In another aspect, the invention provides a method for preventingbacterial attachment or growth on an abiotic surface comprising coatingsaid surface with a peptide capable of inhibiting protein synthesisthrough an interaction at a stem-loop H18 in 16S rRNA of a 30S ribosomalsubunit in an amount sufficient to inhibit the growth of bacteria onsaid abiotic surface. The surface is part of a medical device.Non-limiting examples of a medical device include a syringe, a stent, acatheter, fluid container, a pacemaker, and an implantable pump.

In another aspect, the invention provides peptides comprising consensussequence AMS capable of inhibiting protein synthesis. The peptide iscapable of binding to 30S ribosomal subunit. The binding can involve aninteraction with the stem-loop HIS in 16S rRNA.

In another aspect, the invention discloses peptide selected from Table 1and Table 2 (SEQ ID Nos: 2-33) and analogs and derivatives thereof. Thepeptides can be used to inhibit protein synthesis in prokaryotes, and/oreukaryotes. In some embodiments, the peptides can be attached to amolecule to direct the peptide to a target cell. In other embodiments,the peptide sequences can be used in screening assays and/orligand-displacement assays. For example, the peptides can befluorescently, radioactively, and/or luminescently labeled. The peptidesequences can be coupled to organic groups, fluorescent molecules,radioactive isotopes, other antibiotics, crosslinking agents, and/oroligonucleotides. In yet another embodiment, the peptides can be used assurrogate ligands for high-throughput screening. In some embodiments,the peptides can be used as antimicrobial or antibacterial agents. Inother embodiments, the peptides can be used as anticancer agents. Inother embodiments, the peptides of the present invention can be used aspeptidomimetic drugs. In other embodiments, the peptides can be used aslead compounds to construct libraries of derivatives based on theirsequence. The libraries of derivatives can be used in computationaldocking studies. In other embodiments, the peptides can be used as leadcompounds in structure-based virtual screening of libraries. In yetanother embodiment, the peptides can be used as surrogate ligands forhigh-throughput screening.

In another aspect, the invention provides methods of determining theaffinity of a test compound for a ribosomal subunit comprising the stepsof incubating a ribosomal subunit with one or more detectably labeledpeptides of Tables 1 and 2 (SEQ ID Nos: 2-33); detecting the labeledpeptide bound to the ribosomal subunit; contacting a test compound withthe ribosomal subunit in the presence of the one or more labeledpeptides of Tables 1 and 2; and determining binding of the test compoundto said ribosomal subunit by measuring the change in the detectablelabel. In yet another aspect, the invention provides a method ofidentifying test compounds with binding affinity to a ribosomal subunit,comprising contacting a ribosomal subunit with a detectably labeledpeptide of the present invention to form a labeled complex and measuringa signal from said labeled complex to determine a baseline signal;incubating a ribosomal subunit with a test compound to form a mixture;adding the detectably labeled peptide to said mixture of the ribosomalsubunit and the test compound; measuring the signal from said mixture;comparing the baseline signal with the signal from the mixture, wherebya modulation in said signal indicates that the test compound binds tothe ribosomal subunit. The detectable label can be selected from thelist consisting of a fluorescent label, a chemiluminescent label, acolorimetric label, an enzymatic marker, and a radioactive isotope. Forexample, the fluorescent label can be selected from the non-limitinglist consisting of dansyl, fluorescein, Oregon green, rhodamine,tetra-methyl rhodamine, Texas-red, phycoerythrin, BODIPY fluorophore,and Eu³⁺. The methods can determine affinity for the stem-loop H18 in16S rRNA of a 30S ribosomal subunit.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is schematic of the RNA sequence (SEQ ID NO. 1) and secondarystructure of the stem-loop H18 RNA/DNA hybrid from the 16S rRNA of B.anthracis;

FIG. 2A is a schematic diagram of an exemplary selection scheme used foraffinity selection;

FIG. 2B is a schematic of an exemplary selection scheme used foraffinity selection;

FIG. 3 is a bar graph comparing the peptides tested in the E. colicell-free transcription-translation system for the inhibition orstimulation of synthesis of the firefly luciferase at 100 μM peptideconcentration;

FIG. 4A is a graph showing the inhibition of firefly luciferasesynthesis by BLS15 (SEQ ID NO. 2) in an E. coli cell-freetranscription-translation system;

FIG. 4B is a graph showing the inhibition of firefly luciferasesynthesis by BLS 18 (SEQ ID NO. 3) in an E. coli cell-freetranscription-translation system;

FIG. 5A is a graph showing the inhibition of firefly luciferasesynthesis by BLS15 (SEQ ID NO. 2) in an eukaryotic cell-free translationsystem;

FIG. 5B is a graph showing the inhibition of firefly luciferasesynthesis by BLS 18 (SEQ ID NO. 3) in an eukaryotic cell-freetranslation system; and

FIG. 6 is a graph showing the synergistic effect of BLS 15 (SEQ ID NO.2) with streptomycin.

DETAILED DESCRIPTION OF THE INVENTION

The terms used in this invention are, in general, expected to adhere tostandard definitions generally accepted by those having ordinary skillin the art of microbiology. A few exceptions, as listed below, have beenfurther defined within the scope of the present invention.

The term “antimicrobial compound” as used herein, refers to any compoundthat reduces microbial growth. Such a compound includes, but is notlimited to, polypeptides, proteins, small molecular weight organicmolecules, hormones, and the like. The term “antimicrobial compound” isart-recognized and is intended to include a compound which inhibits theproliferation or viability of a microbe which is undesirable and/orwhich disrupts a microbial cell. The language further includessignificant diminishment of a biological activity which is undesirableand associated with the microbe, such that a subject would not bedetrimentally affected by the microbe. Examples include antibiotics,biocides, antibacterial compounds.

The term “test compound” as used herein, refers to any compoundsuspected of having a capability of interacting with a ribosomal subunitthat results in antimicrobial activity. For example, a “test compound”includes, but is not limited to, protein translation inhibitors,peptides, metabolic inhibitors, polypeptides, small molecular weightorganic molecules, and the like. The term “compound” is art-recognizedand includes compounds being tested for antimicrobial activity. Thecompound can be designed to incorporate a moiety known to interact witha ribosomal subunit or can be selected from a library of diversecompounds, e.g., based on a desired activity, e.g., random drugscreening based on a desired activity. Preferably, the compound of thepresent invention is a small molecule. Examples of compounds of thepresent invention include peptides listed in Tables 1 and 2.

The term “ribosomal protein” as used herein, refers to any polypeptidethat is assembled into a functional ribosome. For example, abacterialribosomal protein includes, but is not limited to, S12, L16, L35, orL36.

The term “ribosomal nucleic acid” as used herein, refers to any nucleicacid that is assembled into a functional ribosome. For example, abacterial nucleic acid includes, but is not limited to, 5S, 16S, 23S,30S, 50S rRNA.

The term “peptide” as used herein, refers to a condensed polymer ofamino acids within which each amino acid is joined by a peptide bond tothe immediately preceding and to the immediately subsequent amino acidin the chain. A peptide comprises a first amino acid, generally referredto as the amino-terminal amino acid, and a last amino acid, generallyreferred to as the carboxyl terminal amino acid. Peptides include, butare not limited to, linear, branched, or cyclic forms. Generally, apeptide comprises a mixture of less than 50, less than 40, less than 30,preferably less than 20, most preferably less than 15 naturallyoccurring or synthetic amino acids. The amino acid may be covalentlymodified.

The term “protein translation” as used herein, refers to the processwhereby free amino acids are enzymatically condensed into peptidergicpolymers, thus forming polypeptides and proteins. The formation ofpeptide bonds is facilitated by intracellular structures (ribosomes)that provide support and enzymatic control for the polymer synthesis.

The term “small molecular weight organic molecule” refers to anyprotease-resistant compound capable of interacting with a polypeptide orprotein. For example, a small molecular weight organic molecule mayrange between approximately 5-1,500 daltons, preferably between 100-750daltons, and more preferably between 250-500 daltons.

The term “infectious disease”” is meant to include disorders caused byone or more species of bacteria, viruses, fungi, and protozoans, speciesof which that are disease-producing organisms collectively referred toas “pathogens.” The term “fungi” is meant to include the yeasts. In thisinvention, pathogens are exemplified, but not limited to, Gram-positivebacteria such as Actinomyces bovis, Enterococcus fecalis, Hemophiluspneumoniae, Listeria monocytogenes, Mycobacterium tuberculosis, M.leprae, M smegmatis, Proprionibacterium acnes, Sarcina ventriculi,Staphylococcus aureus, S. epidermis, S. intermedias, Streptococcushemolyticus, & pneumoniae; Gram-negative bacteria such as Campylobacterfetus, Erwinia carotovora, Flavobacterium meningosepticum, Helicobacterpylori, Hemophilus pneumoniae, H. influenzae, Klebsiella pneumonia,Neisseria gonorrhoeae, Pseudomonas aeruginosa, Shigella dysenteria,Salmonella typhi, S. paratyphi, Yersinia pestis, Escherichia coliserotype 0157, and Chlamydia species, Helicobacter species; viruses suchas HIV-1, -2, and -3, HSV-I and -II, non-A non-B non-C hepatitis virus,pox viruses, rabies viruses, and Newcastle disease virus; fungi such asCandida albicans, C. tropicalis, C. krusei, C. pseudotropicalis, C.parapsilosis, C. quillermondii, C. stellatoidea, Aspergillus fumigatus,A. niger, A. nidulans, A. flavus, A. terreus, Absidia corymbifera, A.ramosa, Cryptococcus neoforms, Histoplasma capsulatum, Coccidioidesimmitis, Pneumocystis carinii, Rhizopus arrhizus, R. oryzae, Mucorpusillus and other fungi; and protozoa such as Entamoeba histolytica,Entamoeba coli, Giardia lamblia, G. intestinalis, Eimeria sp.,Toxoplasma sp., Cryptosporidium parvum, C. muris, C. baileyi, C.meleagridis, C. wrairi, and C. nosarum. Obtaining unique epitopes fromthese organisms by screening proteins and by assaying peptides in vitroare commonly known to those skilled in the art. In one aspect, thescreening methods of the present invention can be used to identifypeptides and/or target compounds with binding affinity to one or more ofthese organisms.

The term “antimicrobial compound” is art-recognized and is intended toinclude a compound which inhibits the proliferation or viability of amicrobe which is undesirable and/or which disrupts a microbial cell. Thelanguage further includes diminishment of an activity which isundesirable and associated with the microbe. Examples includeantibiotics, biocides, antibacterial compounds.

The term “antibiotics” is art recognized and includes antimicrobialagents synthesized by an organism in nature and isolated from thisnatural source, and chemically synthesized antibiotics. The termincludes but is not limited to: polyether ionophore such as monensin andnigericin; macrolide antibiotics such as erythromycin and tylosin;aminoglycoside antibiotics such as streptomycin and kanamycin; β-lactamantibiotics such as penicillin and cephalosporin; and polypeptideantibiotics such as subtilisin and neosporin. Semi-synthetic derivativesof antibiotics, and antibiotics produced by chemical methods are alsoencompassed by this term. Non-limiting examples of antibiotics that canbe used in combination with the peptides of the present invention arelisted herein.

Chemically-derived antimicrobial agents such as isoniazid, trimethoprim,quinolines, and sulfa drugs are considered antibacterial drugs, althoughthe term antibiotic has been applied to these. These agents andantibiotics have specific cellular targets for which binding andinhibition by the agent or antibiotic can be measured. For example,erythromycin, streptomycin and kanamycin inhibit specific proteinsinvolved in bacterial ribosomal activity; penicillin and cephalosporininhibit enzymes of cell wall synthesis; and rifampicin inhibits the βsubunit of bacterial RNA polymerase. It is within the scope of thescreens of the present invention to include compounds derived fromnatural products and compounds that are chemically synthesized.

The term “subject” refers to any living organism in which an immuneresponse is elicited. The term refers to a living animal or human inneed of treatment for, or susceptible to, a condition involving anunwanted or undesirable microorganism, e.g., a particular treatment forhaving an unwanted pathogenic cell as defined below. The term subjectincludes, but is not limited to, humans, nonhuman primates such aschimpanzees and other apes and monkey species; farm animals such ascattle, sheep, pigs, goats and horses; domestic mammals such as dogs andcats; laboratory animals including rodents such as mice, rats and guineapigs, and the like. The term does not denote a particular age or sex.Thus, adult and newborn subjects, as well as fetuses, whether male orfemale, are intended to be covered. In preferred embodiments, thesubject is a mammal, including humans and non-human mammals. In the mostpreferred embodiment, the subject is a human. The term “subject” doesnot preclude individuals that are entirely normal with respect to havingan unwanted pathogen or normal in all respects. The subject may formerlyhave been treated with antibiotic or antimicrobial therapy, and may beunder treatment, or have been treated by antibiotic or antimicrobialtherapy in the past.

The term “patient,” as used herein, refers to a human subject who haspresented at a clinical setting with a particular symptom or symptomssuggesting one or more diagnoses of having an infectious disease, orhaving the presence of an unwanted microbial cell. A patient's diagnosiscan alter during the course of disease progression, such as developmentof further disease symptoms, or remission of the disease, eitherspontaneously or during the course of a therapeutic regimen ortreatment. Thus, the term “diagnosis” does not preclude differentearlier or later diagnoses for any particular patient or subject. Theterm “prognosis” refers to assessment for a subject or patient of aprobability of developing a condition associated with or otherwiseindicated by presence of one or more unwanted pathogenic cells in thepatient.

The invention is described in more detail in the following subsections:

I. Ribosome

The ribosome is the cellular machinery in charge of protein synthesis.Many antibiotics target the ribosome by interfering with the mechanismof protein synthesis. The widespread use of antibiotics has propelledthe outburst of bacteria resistant to antibiotics. In one aspect, theinvention identifies new ribosomal sites for antibiotic targeting.

Most of the antibacterial agents that inhibit protein synthesis interactwith the bacterial ribosome. The differences between the composition ofbacterial and mammalian ribosomes give these compounds theirselectivity. For example, aminoglycosides are a group of structurallyrelated compounds containing three linked hexose sugars. Aminoglycosidesexert a bactericidal effect by binding irreversibly to the 30S subunitof the bacterial ribosomes and blocking initiation of protein synthesis.Macrolides and lincosamides, although structurally different, are twotypes of antibiotics that bind specifically to the 50S portion of thebacterial ribosome. Chloramphenicol also binds irreversibly to the 50Sportion of the bacterial ribosome at a site close but not identical tothe sites binding the macrolides and lincosamides. Tetracyclinesinteract reversibly with the bacterial 30S ribosomal subunit, blockingthe binding of aminoacyl tRNA to the mRNA-ribosome complex. Thismechanism is markedly different from that of the aminoglycosides, whichalso bind to the 30S subunit.

Translation of the genetic code occurs on the ribosome, a largenucleoprotein complex that consists of two subunits. In bacteria, thetwo subunits are denoted 30S and 50S. The 50S subunit contains thecatalytic site of peptidyl transferase activity, while the 30S subunitplays a crucial role in decoding messenger RNA. The 30S ribosomalsubunit is a major target for antibiotics. The ribosome is a usefultarget for antibiotics since the structure of the 30S is widelyconserved between prokaryotes, allowing for broad spectrum antibiotics.However, resistance to current antibiotics is currently a major problemin the field of medicine. There are presently very few new antibioticsavailable which can be used to treat the highly resistant strains ofbacteria such as MRSA (methicilin resistant Staphylococcus aureus) whichare becoming increasingly widespread.

In one aspect, the invention identifies new ribosomal sites forantibiotic targeting. The stem-loop H18 in 16S rRNA from 30S ribosomalsubunit plays a central role in the decoding of proteins, and folds intoa pseudoknot whose disruption is detrimental for translation ofproteins, leading to cell death. H18 is a very conserved region andmutations are detrimental for function, characteristic that has beenobserved for other critical functional centers of the ribosome, alreadybeing targeted by common antibiotics. H18 is an unexploited site, withpotential for antibiotic action, it has not been targeted before, and noknown antibiotic exists that bind this site. H18 will be a useful sitefor the development of new antibacterials, specially useful forsuperbugs that are resistant in some cases to all antibiotics known.

In one aspect, the invention provides a novel screening technique forselecting peptides with specific binding properties from a large libraryof random peptide motifs. A novel version of phage display is disclosedfor identifying new peptides with binding affinity for new target sitesin the ribosome. In some embodiments, peptides with affinity for atertiary structural motif of the ribosomal RNA can be selected. Inpreferred embodiments, peptides with affinity for the stem-loop H18 ofthe 16S rRNA small subunit are identified. For example, peptides can beidentified with a binding affinity greater than 30%, greater than 50%,greater than 60%, greater than 75%, and greater than 85%. In preferredembodiments, the peptides can inhibit protein synthesis. In someembodiments, the peptides can be used in antimicrobial and/orantibacterial compositions. In other embodiments, the peptides can beused to inhibit cancer cell proliferation. The peptides can be coupledto moieties, such that the peptides are specifically directed to adesired target cell. In yet other embodiments, the peptides can be usedin screening assays. The peptides can be coupled to fluorescentmolecules, radioactive isotopes, other antibiotics, crosslinking agents,and/or oligonucleotides.

In one aspect of the present invention, a novel methodology wasdeveloped to screen a library of peptides in order to find peptides withprotein synthesis inhibiting properties. In some examples, the methodscan be used to identify peptides that can bind to a prokaryoticribosomal subunit and antibacterial properties. For example, peptideswith binding affinity for H18. By means of this methodology, peptideinhibitors of protein synthesis were identified. The selected peptidesshow inhibitory activity in prokaryotic systems. In some embodiments,the peptides of the invention show protein synthesis inhibition ineukaryotes. These peptides can be used, for example, as anticanceragents. In some embodiments, the peptides can be attached to moietycapable of directing the peptide to a desired target cell.

In some other embodiments, the peptides can be used to stimulate proteinsynthesis. Stimulation of protein synthesis characteristic useful toenhance the yield of protein synthesis in cases in which the efficiencyof synthesis is poor. The identified peptides exhibit synergy with otherantibiotics, having the potential to be used in combination therapies orcan be coupled with other antibiotics to improve potency. The peptidesof the present invention can be used as leads to screen libraries ofcompounds targeting the new H18 site, helping in the task of finding newantibacterials with binding affinities for H18, and having thefunctional properties of being inhibitors of protein synthesis. Thepeptides of the present invention can be also used in detailedstructure/activity relationship analysis to generate derivatives and newclasses of antibacterials.

In another aspect, a method was developed to examine the binding of thediscovered peptides to the ribosome by tagging the peptides tofluorescent molecules.

II. Novel Selection Technique

In one aspect of the invention, a phage display technique is disclosedfor identifying new peptide ligands with binding affinity to new targetsites in the ribosome. Phage display is a system in which a protein isdisplayed on the surface of a phage as a fusion with one of the coatproteins of the virus. The DNA that encodes this protein is housedwithin the virion. By cloning large numbers of DNA sequences into thephage, display libraries are produced with a repertoire of many billionsof unique displayed proteins. Phage display can be used as a screeningtechnique for selecting peptides with specific binding properties from alarge library of random peptide motifs. Advantages of using phagedisplay over other approaches for drug discovery are that the scale ofthe screening is much greater than when using chemical libraries,billions of phage are screened at the same time in comparison tothousands of compounds screened one by one; the affinity of the peptideover the target can be modulated, the screening can be done underspecific conditions that will improve binding specificity, peptides canbe selected by their binding and functional capabilities, and the cDNAis in the phage and thus no separate cloning step is required.

Ribosomal RNA folds into tertiary structural motifs that are excellenttarget for peptides, in particular the stem-loop H18 of the 16S rRNAsmall subunit is a new potential target for antibiotics. The stem-loopH18 RNA forms the higher order structure of a pseudoknot, generating abinding pocket capable of accommodating ligands such as peptides. Smallpeptides are unstructured in solution, and adopt a distinct fold whencomplexed with RNA, originating a conformational change in the RNA uponbinding. The stem-loop H18 RNA is important for function in thesynthesis of proteins and disruption of the secondary structure inducedby a conformational change will lead to the inhibition of proteinsynthesis and as a consequence cell death. In some embodiments, the newtarget site can be combined with currently available drug targets suchthat new ligand peptides can be identified. This technique will delaythe appearance of resistance for the newly discovered antibiotics.

Peptides with antibacterial activity targeting the stem-loop H18 RNAwere identified using a newly developed protocol to select for phagedisplaying peptides that selectively bind the H18 RNA, site that has notbeen previously used for antibiotic screening. A 59-nt RNA-DNA constructrepresenting the stem-loop H18 of Bacillus anthracis small ribosomalsubunit (FIG. 1) was designed. Phage display technology faces theproblem of phage having high affinity for plastics, wells, streptavidinand other materials used during the screening, we solved this problem asfollows: first a counter selection was done against materials used forthe affinity selection, and by optimization of the number and stringencyof washes to remove unbound phage after each cycle of selection; second,the selection was done using a novel strategy in which the RNA targetused for selection was a extended target molecule (e.g. hybrid RNA/DNAor hybrid RNA/organic molecule) and the extended target molecule (e.g.DNA or an organic molecule) was tagged with an affinity linker (e.g.biotin molecule), this system allows for the specific selection of phagebinding to the RNA target; third, the isolation of phage bound to theRNA target was done with immobilized surfaces (e.g., magnetic beads ormicrotiter plates) coated with a binding partner of an affinity linkerof the extended target molecule, and only phage bound to the RNA targetare pulled out from solution; fourth, unbound phage are removed by aseries of washes in the presence of a detergent that will remove anyphage bound nonspecifically. The advantage of this novel design is theability of releasing specific phage-RNA complexes obtained from theinitial nonspecific pool of 2×10¹¹ phage. The specific phage-RNAcomplexes are released from the capture system (magnetic beads or wells)by the use of the enzyme (e.g., deoxyribonuclease I), that will digestonly and specifically the extended target molecule (e.g. DNA portion ofan RNA/DNA hybrid). Therefore the phage with affinity to the H18 RNA areliberated and ready to be used for further selection steps.

A general description of a screening method of the present invention isgraphically depicted in FIGS. 2A and 2B and described below. Example 2demonstrates that the methods of the present invention can be used toidentify test compounds, i.e., peptides with affinity for a ribosomalsubunit. The affinity selection methodology was designed and developedusing the two capture formats of magnetic beads and microtiter plates,and two elution systems including nonspecific elution, and specificelution by enzymatic digestion of the specifically designed poly-dA tailof H18 RNA with DNase I. H18 RNA at a concentration of 100 nM wasallowed to interact with the phage library in solution in the presenceof tRNA (2 μg/μL). The RNA-binding phage were captured using eithercapture format, and biotin was added to a final concentration of 0.1 mMin order to displace any streptavidin-binding phage from the solidsupport. Unbound phage were washed from the solid support with washbuffer supplemented with tween-20. The stringency of washes wasincreased for the magnetic beads capture system, by increasing theconcentration of tween-20 from 0.1% to 0.5% after the first round ofselection. The stringency of washes for the microtiter well plate systemwas kept constant throughout the rounds of selection by using aconcentration of tween-20 of 0.05%. The bound phage were released fromthe solid support using an acidic buffer as a nonspecific eluant. Afterfour rounds of selection the sequences of the phage-displayed peptideswere obtained by sequencing of the phage DNA.

An additional round of selection was performed using the amplifiedeluate from the fourth round of magnetic beads selection. The boundphage were eluted by using an enzyme capable of cleaving the extentionmoiety. A separate affinity selection was carried out using animmobilized capture system and eluting the bound phage specifically withan enzyme. An overview of an example of the selection process is shownin FIGS. 2A and B.

II. Synergism and Combinations with Antibiotics

Currently, there are several types of antibiotic compounds in useagainst bacterial pathogens, and these compounds act through a varietyof anti-bacterial mechanisms. In one aspect, the identified peptides canbe used in combination with other antibiotics. In some embodiment, newribosomal sites can be targeted in combination with new or currentantibacterial or antimicrobial agents. Combination therapy can helpdelay new resistance mechanisms.

Drug combinations are known to reduce the dosages required, and in somecases, produce synergistic effects. Thus, in order to increase theeffectiveness of the peptide therapies described herein, it may bedesirable to combine these compositions with other agents effective inthe treatment of bacterial infections, such as antibiotics.

An “antibiotic” agent is capable of negatively affecting bacterialgrowth in a subject, for example, by killing bacterial cells, reducingthe growth rate of bacterial cells, or otherwise increasing the qualityof life of the afflicted subject. This process may involve contactingthe cells with the peptide the agent(s) at the same time. This may beachieved by contacting the cell with a single composition orpharmacological formulation that includes both the peptide and theantibiotic, or by contacting the cell with two distinct compositions orformulations, at the same time, wherein one composition includes thepeptide and the other includes the second agent(s).

Alternatively, the peptide therapy may precede or follow the other agenttreatment by intervals ranging from minutes to weeks. In embodimentswhere the other agent and peptide are applied separately to the cell,one would generally ensure that a significant period of time did notexpire between the time of each delivery, such that the agent andpeptide would still be able to exert an advantageously combined effecton the cell. In such instances, it is contemplated that one may contactthe cell with both modalities within about 1224 h of each other and,more preferably, within about 612 h of each other. In some situations,it may be desirable to extend the time period for treatmentsignificantly, however, where several days (2, 3, 4, 5, 6 or 7) toseveral weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respectiveadministrations.

Administration of the peptides of the present invention to a patientwill follow general protocols for the administration of antibiotics,taking into account the toxicity, if any. It is expected that thetreatment cycles would be repeated as necessary.

Various classes of antibiotics that can also be used in combination withthe invention are described below. For example, beta-lactam antibiotics,such as penicillin and cephalosporin, act to inhibit the final step inpeptidoglycan synthesis. Glycopeptide antibiotics, including vancomycinand teichoplanin, inhibit both transglycosylation and transpeptidationof muramyl-pentapeptide, again interfering with peptidoglycan synthesis.Other well-known antibiotics include the quinolones, which inhibitbacterial DNA replication, inhibitors of bacterial RNA polymerase, suchas rifampin, and inhibitors of enzymes in the pathway for production oftetrahydrofolate, including the sulfonamides.

Antibacterial agents, like all anti-microbial drugs, are directedagainst unique targets not present in mammalian cells. The goal is tolimit toxicity to the host and maximize chemotherapeutic activityaffecting invading microbes only. One major difference between bacterialand mammalian cells in the presence in bacteria of a rigid wall externalto the cell membrane. The wall protects bacterial cells from osmoticrupture because of the difference between the hyperosmolar (up to 20atm) cell interior and the usually isosmolar or hyposmolar hostenvironment. In both gram-positive and gram-negative bacteria,peptidoglycan, a large, covalently linked sacculus that surrounds thebacterium, is the structure that confers cell wall rigidity andresistance to osmotic lysis. In gram positive bacteria, peptidoglycan isthe only later external to the cell membrane and is thick (20 to 80 nm);while in gram-negative bacteria the peptidoglycan layer is thin (1 nm)and is protected by an outer membrane. Chemotherapeutic agents directedat any stage of the synthesis, export, assembly, or cross-linking ofpeptidoglycan inhibit bacterial cell growth and, in most cases, lead tocell death.

Bacitracin, a cyclic peptide antibiotic, inhibits the conversion to itsactive form of the lipid carrier that moves the water-solublecytoplasmic peptidoglycan subunits through the cell membrane to the cellexterior. Cell wall subunits accumulate in the cytoplasm and can beadded to the growing peptidoglycan chain.

Cyclopeptides, (such as vancomycin and teichoplanin) are high molecularweight antibiotics that bind to the terminal D-alanine-D-alaninecomponent of the stem peptide when the subunits are external to the cellmembrane and still linked to the lipid carrier. This binding stericallyinhibits the addition of sub units to the peptidoglycan backbone.

β-Lactam antibiotics, such as penicillins, cephalosporins, carbapenems,and monobactams, characterized by a four-membered β-Lactam ring, preventthe cross-linking reaction called transpeptidation. Energy for attachinga peptide cross-bridge from the stem peptide of one peptidoglycansubunit to another is derived from the cleavage of a terminal D-alanineresidue from the subunit stem peptide. The β-Lactam ring of theantibiotic forms an irreversible covalent acyl bond with thetranspeptidase enzyme, preventing the cross-linking reaction.Transpeptidases and similar enzymes involved in cross-linking are calledpenicillin-binding proteins because they have active sites that bindβ-Lactam antibiotics.

Virtually all the antibiotics that inhibit bacterial cell wall synthesisare bactericidal, eventually resulting in the cell's death due toosmotic lysis. However, much of the loss of cell wall integrityfollowing treatment with cell-active agents is due to the bacteria's owncell wall-remodeling enzymes (autolysins) that cleave peptidoglycanbonds in the normal course of cell growth. Autolysis without normal cellwall repair results in weakness and eventual cell death.

Some classes of antibiotics act at the level of protein synthesis.Notable among these are the aminoglycosides, such as kanamycin andgentamycin. This class of compounds targets the bacterial 30S ribosomesubunit, preventing the association with the 50S subunit to formfunctional ribosomes. Tetracyclines, another important class ofantibiotics, also target the 30S ribosome subunit, acting by preventingalignment of aminoacylated tRNA's with the corresponding mRNA codon.Macrolides and lincosamides, another class of antibiotics, inhibitbacterial synthesis by binding to the 50S ribosome subunit, andinhibiting peptide elongation or preventing ribosome translocation.

The antimetabolites are all synthetic compounds that interfere withbacterial synthesis of folic acid. Products of the folic acid synthesispathway function as coenzymes for the one-carbon transfer reactions thatare essential for the synthesis of thymidine, all purines, and severalamino acids. Inhibition of folate synthesis leads to cessation of cellgrowth and in some cases, to bacterial cell death. The principalantibacterial antimetabolites are sulfonamides and trimethoprim.

Numerous additional antibacterial compounds have disparate effects onnucleic acids. The quinolones, including nalidixic acid and itsfluorinated derivatives, are synthetic compounds that inhibit theactivity of the A subunit of the bacterial enzyme DNA gyrase, which isresponsible for negative supercoiling of DNA during replication in theintact cell. The antibiotic novobiocin also interferes with the activityof DNA gyrase, but it interferes with the B subunit. Rifampin, usedprimarily as an antituberculosis agent, binds tightly to bacterialDNA-dependent RNA polymerase, thus inhibiting transcription of DNA intoRNA, and nitrofurantoin, a synthetic compound, causes DNA damage, beingreduced by a bacterial enzyme to highly reactive, short-livedintermediates that are thought to cause DNA strand breakage.

Still other compounds cause alternation of cell membrane permeability.The polymyxins behave as cationic, surface-active compounds that disruptthe permeability of both the outer and the cytoplasmic membranes ofgram-negative bacteria. Gramicidin A, on the other hand, acts as anionophore, forming pores or channels in lipid bilayers.

One major and important class of genes consists of those bacterial genesthat are essential for growth or viability of a bacterium. Becauseuseful conventional antibiotics, such as those described above, areknown to act by interfering with the products of essential genes, it islikely that the discovery of new essential gene products will have asignificant impact on efforts to develop novel antimicrobial drugs.

Conditional mutations such as temperature or suppressor sensitivemutations have been used in the past to identify some of the essentialgenes in bacteria. However, not all essential genes can be identified bythese types of mutations. The limitation is due to the fact that theseconditional mutations must occur at a specific codon of the genes inorder to alter the coded amino acid of the protein. Therefore, theoccurrence of mutants with these phenotypes is expected to be low.Moreover, not all of the gene can be converted to conditional mutations;there may be no codon causing these mutations in their nucleotidesequences.

Essential gene products have been traditionally identified through theisolation of conditional lethal mutants, or by transposon mutagenesis inthe presence of a complementing wild type allele (balanced lethality).However, such approaches are laborious, as they require identification,purification, and study of individual mutant strains. These methods arealso limited to species with well-developed systems for geneticmanipulation and, therefore, cannot be readily applied to many of thepotentially dangerous microorganisms whose genomes have recently beensequenced.

For example, conditional mutations such as temperature or suppressorsensitive mutations have been used in the past to identify some of theessential genes in bacteria. However, not all of the essential genes canbe identified by these types of mutations because the conditionalmutation must occur at a specific codon of the genes in order to alterthe coded amino acid of the protein. Therefore, the occurrence ofmutants with these phenotypes is expected to be low. Moreover, not allof the gene can be converted to conditional mutations; there may be nocodon causing the conditional mutation in the nucleotide sequences ofthe bacterial genome.

III. Antimicrobial Compound Compositions and Uses

The invention provides pharmaceutically acceptable compositions whichinclude a therapeutically-effective amount or dose of an antimicrobialcompound, e.g., peptides from Tables 1 and 2, and one or morepharmaceutically acceptable carriers (additives) and/or diluents. Acomposition can also include a second antimicrobial agent, e.g., aninhibitor of an efflux pump. In some embodiments, the peptides of thepresent invention can be coupled to moieties, such that the peptides canbe directed to a target cell. The invention identifies a method ofinhibiting protein synthesis in a cell comprising administering acompound capable interacting with a ribosome at a stem-loop H18 in 16SrRNA of a 30S ribosomal subunit.

In preferred embodiments compounds of the invention can be used toinhibit the growth of an unwanted organism, e.g., an infectious,pathogenic organism or an organism that causes spoilage or biofouling,by contacting the organism with the compound. The compound can beapplied prior infection by the organism to prevent a subject frombecoming infected. For example, the compounds can be used for cleaningsurfaces, e.g., counter tops, instruments, or the skin of the subject,to inhibit the growth of the organism and reduce the possibility of thesubject actually becoming infected with one of the organisms.

Treating or treatment of a state characterized by the presence of anunwanted cell, e.g., an unwanted pathogenic cell, e.g., an unwantedbacterium, is intended to include the alleviation of or diminishment ofat least one symptom, for example, fever or inflammation, typicallyassociated with the state. The treatment also includes alleviation ordiminishment of more than one symptom. Preferably, the treatment cures,e.g., substantially eliminates, the symptoms associated with the state.

The terms “therapeutically effective dose” or “therapeutically effectiveamount” of a compound or peptide described herein, is that amountnecessary or sufficient to perform its intended function, e.g., on asurface or on or within a subject, e.g., to eradicate or inhibit growthof an unwanted pathogen, e.g., microorganism. The therapeuticallyeffective amount can vary depending on such factors as the species orstrain of the pathogen, the amount of the pathogen to be inhibited antthe manner in which the compound is to be used. One of ordinary skill inthe art would be able to study the aforementioned factors and make adetermination regarding the effective amount of the compound requiredwithout undue experimentation. For administration, one of ordinary skillin the art would be able to determine such amounts based on such factorsas the subject's size, the severity of the subject's symptoms, and theparticular composition or route of administration selected. An in vitroor in vivo assay can be here used to determine an “effective amount” ofthe compounds described herein to achieve inhibition of growth orproliferation of the cell by binding and inhibiting the specific target.

A “therapeutically effective dosage” is a dosage of a compound thatpreferably inhibits growth of an unwanted pathogenic cell, or destroyscell viability, by at least about 50%, more preferably by at least about80%, even more preferably by at least about 90%, and still morepreferably by at least about 95% relative to the absence of thecompound. The ability of a compound to inhibit or kill infectiousdisease cells can be evaluated in an in vitro inhibitory concentrationassay, or, e.g., an animal model system predictive of efficacy ininfectious diseases. Alternatively, this property of a compound can beevaluated by examining the ability of the compound to inhibit in vitroby using assays well-known to the skilled practitioner. Assays includethe of effect on viability of the test pathogenic cell, by assay ofquantity of “colony forming units” (cfu), in the presence and absence ofthe compound; assay of capability to carry out a physiological process,such as cellular uptake of a metabolite; assay of uptake andincorporation of a metabolite into a macromolecule, such as a nucleicacid or protein; each assay conducted in the presence of a range ofconcentrations and in the absence of the compound. For compounds havinga known specific target, the effective dosage to inhibit the activity ofthat target, such as an enzyme, can be assessed using isolated targetmaterial. In addition, the antimicrobial agents and compounds of theinvention can also be used, for example, in antimicrobial soap ordetergent preparations.

IV. Pharmaceutical Compositions

As described in detail below, the pharmaceutical compositions can beformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), tablets,boluses, powders, granules, pastes; (2) parenteral administration, forexample, by subcutaneous, intramuscular or intravenous injection as, forexample, a sterile solution or suspension; (3) topical application, forexample, as a cream, ointment or spray applied to the skin; (4)intravaginally or intrarectally, for example, as a pessary, cream, foam,or suppository; or (5) aerosol, for example, as an aqueous aerosol,liposomal preparation or solid particles containing the compound.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the antimicrobial agentsor compounds of the invention from one organ, or portion of the body, toanother organ, or portion of the body without affecting its biologicaleffect. Each carrier should be “acceptable” in the sense of beingcompatible with the other ingredients of the composition and notinjurious to the subject. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalcompositions. Proper fluidity can be maintained, for example, by the useof coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Pharmaceutical compositions of the present invention may be administeredto epithelial surfaces of the body orally, parenterally, topically,rectally, nasally, intravaginally, intracistemally. They are of coursegiven by forms suitable for each administration route. For example, theyare administered in tablets or capsule form, by injection, inhalation,eye lotion, ointment, etc., administration by injection, infusion orinhalation; topical by lotion or ointment; and rectal or vaginalsuppositories.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrastemal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a sucrose octasulfate and/or anantibacterial or a contraceptive agent, drug or other material otherthan directly into the central nervous system, such that it enters thesubject's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

In some methods, the compositions of the invention can be topicallyadministered to any epithelial surface. An “epithelial surface”according to this invention is defined as an area of tissue that coversexternal surfaces of a body, or which and lines hollow structuresincluding, but not limited to, cutaneous and mucosal surfaces. Suchepithelial surfaces include oral, pharyngeal, esophageal, pulmonary,ocular, aural, nasal, buccal, lingual, vaginal, cervical, genitourinary,alimentary, and anorectal surfaces.

Compositions can be formulated in a variety of conventional formsemployed for topical administration. These include, for example,semi-solid and liquid dosage forms, such as liquid solutions orsuspensions, suppositories, douches, enemas, gels, creams, emulsions,lotions, slurries, powders, sprays, lipsticks, foams, pastes,toothpastes, ointments, salves, balms, douches, drops, troches, chewinggums, lozenges, mouthwashes, rinses.

Conventionally used carriers for topical applications include pectin,gelatin and derivatives thereof, polylactic acid or polyglycolic acidpolymers or copolymers thereof, cellulose derivatives such as methylcellulose, carboxymethyl cellulose, or oxidized cellulose, guar gum,acacia gum, karaya gum, tragacanth gum, bentonite, agar, carbomer,bladderwrack, ceratonia, dextran and derivatives thereof, ghatti gum,hectorite, ispaghula husk, polyvinypyrrolidone, silica and derivativesthereof, xanthan gum, kaolin, talc, starch and derivatives thereof,paraffin, water, vegetable and animal oils, polyethylene, polyethyleneoxide, polyethylene glycol, polypropylene glycol, glycerol, ethanol,propanol, propylene glycol (glycols, alcohols), fixed oils, sodium,potassium, aluminum, magnesium or calcium salts (such as chloride,carbonate, bicarbonate, citrate, gluconate, lactate, acetate, gluceptateor tartrate).

Such compositions can be particularly useful, for example, for treatmentor prevention of an unwanted celi, e.g., vaginal Neisseria gonorrhea, orinfections of the oral cavity, including cold sores, infections of eye,the skin, or the lower intestinal tract. Standard composition strategiesfor topical agents can be applied to the antimicrobial compounds, e.g.,compounds of the present invention, i.e., peptides of Tables 1 and 2 ora pharmaceutically acceptable salt thereof in order to enhance thepersistence and residence time of the drug, and to improve theprophylactic efficacy achieved.

For topical application to be used in the lower intestinal tract orvaginally, a rectal suppository, a suitable enema, a gel, an ointment, asolution, a suspension or an insert can be used. Topical transdermalpatches may also be used. Transdermal patches have the added advantageof providing controlled delivery of the compositions of the invention tothe body. Such dosage forms can be made by dissolving or dispersing theagent in the proper medium.

Compositions of the invention can be administered in the form ofsuppositories for rectal or vaginal administration. These can beprepared by mixing the agent with a suitable non-irritating carrierwhich is solid at room temperature but liquid at rectal temperature andtherefore will melt in the rectum or vagina to release the drug. Suchmaterials include cocoa butter, beeswax, polyethylene glycols, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active agent.

Compositions which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams, films, or spraycompositions containing such carriers as are known in the art to beappropriate. The carrier employed in the sucroseoctasulfate/contraceptive agent should be compatible with vaginaladministration and/or coating of contraceptive devices. Combinations canbe in solid, semi-solid and liquid dosage forms, such as diaphragm,jelly, douches, foams, films, ointments, creams, balms, gels, salves,pastes, slurries, vaginal suppositories, sexual lubricants, and coatingsfor devices, such as condoms, contraceptive sponges, cervical caps anddiaphragms.

For ophthalmic applications, the pharmaceutical compositions can beformulated as micronized suspensions in isotonic, pH adjusted sterilesaline, or, preferably, as solutions in isotonic, pH adjusted sterilesaline, either with or without a preservative such as benzylalkoniumchloride. Alternatively, for ophthalmic uses, the compositions can beformulated in an ointment such as petrolatum. Exemplary ophthalmiccompositions include eye ointments, powders, solutions and the like.

Powders and sprays can contain, in addition to sucrose octasulfateand/or antibiotic or contraceptive agent(s), carriers such as lactose,talc, silicic acid, aluminum hydroxide, calcium silicates and polyamidepowder, or mixtures of these substances. Sprays can additionally containcustomary propellants, such as chlorofluorohydrocarbons and volatileunsubstituted hydrocarbons, such as butane and propane.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular compound, buttypically include nonionic surfactants (Tweens, Pluronics, orpolyethylene glycol), innocuous proteins like serum albumin, sorbitanesters, oleic acid, lecithin, amino acids such as glycine, buffers,salts, sugars or sugar alcohols. Aerosols generally are prepared fromisotonic solutions.

Compositions of the invention can also be orally administered in anyorally-acceptable dosage form including, but not limited to, capsules,cachets, pills, tablets, lozenges (using a flavored basis, usuallysucrose and acacia or tragacanth), powders, granules, or as a solutionor a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouth washes and the like, each containinga predetermined amount of sucrose octasulfate and/or antibiotic orcontraceptive agent(s) as an active ingredient. A compound may also beadministered as a bolus, electuary or paste. In the case of tablets fororal use, carriers which are commonly used include lactose and cornstarch. Lubricating agents, such as magnesium stearate, are alsotypically added. For oral administration in a capsule form, usefuldiluents include lactose and dried corn starch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Tablets, and other solid dosage forms, such as dragees, capsules, pillsand granules, may be scored or prepared with coatings and shells, suchas enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active ingredient, the liquid dosage formsmay contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the antimicrobial agent(s) may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Sterile injectable forms of the compositions of this invention can beaqueous or oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. Wetting agents, emulsifiers andlubricants, such as sodium lauryl sulfate and magnesium stearate, aswell as coloring agents, release agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the compositions.

The sterile injectable preparation may also be a sterile injectablesolution or suspension in a nontoxic parenterally-acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose, any bland fixed oil may be employedincluding synthetic mono- or di-glycerides. Fatty acids, such as oleicacid and its glyceride derivatives are useful in the preparation ofinjectables, as are natural pharmaceutically-acceptable oils, such asolive oil or castor oil, especially in their polyoxyethylated versions.These oil solutions or suspensions may also contain a long-chain alcoholdiluent or dispersant, such as Ph. Helv or similar alcohol.

The antimicrobial agent or a pharmaceutically acceptable salt thereofwill represent some percentage of the total dose in other dosage formsin a material forming a combination product, including liquid solutionsor suspensions, suppositories, douches, enemas, gels, creams, emulsions,lotions slurries, soaps, shampoos, detergents, powders, sprays,lipsticks, foams, pastes, toothpastes, ointments, salves, balms,douches, drops, troches, lozenges, mouthwashes, rinses and others.Creams and gels for example, are typically limited by the physicalchemical properties of the delivery medium to concentrations less than20% (e.g., 200 mg/gm). For special uses, far less concentratedpreparations can be prepared, (e.g., lower percent formulations forpediatric applications). For example, the pharmaceutical composition ofthe invention can comprise sucrose octasulfate in an amount of0.001-99%, typically 0.01-75%, more typically 0.1-20%, especially 1-10%by weight of the total preparation. In particular, a preferredconcentration thereof in the preparation is 0.5-50%, especially 0.5-25%,such as 1-10%. It can be suitably applied 1-10 times a day, depending onthe type and severity of the condition to be treated or prevented.

Given the low toxicity of an antimicrobial agent or a pharmaceuticallyacceptable salt thereof over many decades of use as a biocide [W. R.Garnett, Clin. Pharm. 1:307-314 (1982); R. N. Brogden et al., Drugs27:194-209 (1984); D. M. McCarthy, New Eng J. Med., 325:1017-1025(1991), an upper limit for the therapeutically effective dose is not acritical issue. For most forms of compounds of the present invention,i.e., peptides of Tables 1 and 2 the minimum amount present in thematerials forming combinations of this invention that is effective intreating or preventing bacterial disease due to direct interaction withthe organism should produce be less than 0.1 μg/ml, less than 0.5 μg/ml,preferably less than 1 μg/ml, even more preferably less than less than 5μg/ml, and most preferably less than 10 μg/ml.

For prophylactic applications, the pharmaceutical composition of theinvention can be applied prior to physical contact. The timing ofapplication prior to physical contact can be optimized to maximize theprophylactic effectiveness of the compound. The timing of applicationwill vary depending on the mode of administration, the epithelialsurface to which it is applied, the surface area, doses, the stabilityand effectiveness of composition under the pH of the epithelial surface,the frequency of application, e.g., single application or multipleapplications. Preferably, the timing of application can be determinedsuch that a single application of composition is sufficient. One skilledin the art will be able to determine the most appropriate time intervalrequired to maximize prophylactive effectiveness of the compound.

One of ordinary skill in the art can determine and prescribe theeffective amount of the pharmaceutical composition required. Forexample, one could start doses at levels lower than that required inorder to achieve the desired therapeutic effect and gradually increasethe dosage until the desired effect is achieved. In general, a suitabledaily dose of a composition of the invention will be that amount of thecomposition which is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above. It is preferred that administration be intravenous,intracoronary, intramuscular, intraperitoneal, or subcutaneous.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, microbiology, recombinant DNA, and immunology, whichare within the skill of the art. Such techniques are explained fully inthe literature. See, for example, Molecular Cloning A Laboratory Manual,2nd Ed., ed. by Sambrook, J. et al. (Cold Spring Harbor Laboratory Press(1989)); Short Protocols in Molecular Biology, 3rd Ed., ed. by Ausubel,F. et al. (Wiley, NY (1995)); DNA Cloning, Volumes I and II (D. N.Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed. (1984));Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D.Hames & S. J. Higgins eds. (1984)); the treatise, Methods In Enzymology(Academic Press, Inc., N.Y.); Immunochemical Methods In Cell AndMolecular Biology (Mayer and Walker, eds., Academic Press, London(1987)); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weirand C. C. Blackwell, eds. (1986)); and Miller, J. Experiments inMolecular Genetics (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1972)).

The invention provides pharmaceutically acceptable compositions whichinclude a therapeutically-effective amount or dose of an antimicrobialcompound, e.g., peptides from Tables 1 and 2, and one or morepharmaceutically acceptable carriers (additives) and/or diluents. Acomposition can also include a second antimicrobial agent, e.g., aninhibitor of an efflux pump.

One skilled in the art will appreciate further features and advantagesof the invention based on the above-described embodiments. Accordingly,the invention is not to be limited by what has been particularly shownand described, nor by the examples set forth below, except as indicatedby the appended claims. All publications and references cited herein areexpressly incorporated herein by reference in their entirety.

EXAMPLES

This invention is further illustrated by the following examples whichshould not be construed as limiting. The following experiments wereperformed to demonstrate various aspects of the invention.

Example 1 Materials and Methods (i) Materials Target RNA.

The RNA used as a target to select for peptide binding was a 59-ntRNA-DNA construct representing the stem-loop H18 of Bacillus anthracissmall ribosomal subunit, H18 RNA(5′-GGGGCCACGGCUAACUACGUGCCAGCAGCCGCGGUAAUACGUAGGUGGCdAdAdAdAdAdAdAdAdAdABiotin-3′) (Seq. ID. NO: 1). Threeadditional guanosines were added at the 5′ end, and a poly-dA tail of 10nucleotides followed by a biotin molecule was added at the 3′ end. TheRNA-DNA hybrid was obtained from Dharmacon RNA Technologies and purifiedby gel electrophoresis on denaturing (8 M urea) 8% polyacrylamide gels.H18 RNA was renatured in 80 mM HEPES-KOH pH 7.4, the RNA was incubatedat 90° C. for 1 min in a water bath and allowed to cool down to 20° C.for 2 h.

(ii) Screening of Phage-Displayed Peptide Library.

The Ph.D.-7 phage library from New England Biolabs was used, whichcontains a library of 1.28×10⁹ random heptapetides fused to coat proteinpIII of M13 Phage. Two parallel selections were performed, one selectionwas done on microplates coated with streptavidin and another selectionwas done on streptavidin coated magnetic beads.

(iii) Selection on Streptavidin Coated Microplates.

Streptavidin coated plates from Pierce Biotechnology were washed fourtimes with 250 μL of 1× wash buffer for wells (80 mM HEPES-KOH pH 7.4,150 mM KCl, 20 mM MgCl₂, 0.05% tween-20 [v/v]), allowing 3 minincubation between washes. The plates were washed twice with 250 μL of1× binding buffer (80 mM HEPES-KOH pH 7.4, 150 mM KCl, 20 mM MgCl₂).Before starting the selection step a counter selection was made againststreptavidin and plastic. The phage (2×10¹¹ phage) without target RNAwere added to a washed well with 1× binding buffer, and incubated at 4°C. for 1 h. After the counter selection, selection was started bytransferring the phage to a tube containing 80 mM HEPES-KOH pH 7.4, 150mM KCl, 20 mM MgCl₂, 100 nM H18 RNA, 100 μg tRNA, and 40 U RNaseInhibitor. The mixture was incubated at 25° C. for 30 min, and thentransferred to a washed well and incubated at 4° C. for 1 h. In order tocompete out phage binding to streptavidin, biotin at a concentration of0.1 mM was added and the wells were incubated at 4° C. for 5 min. Thesupernatant containing the unbound phage was removed and the wells werewashed four times with 250 μL of 1× wash buffer for wells, and twicewith 1× binding buffer. The H18 RNA-bound phage were eluted by adding100 μL of 0.2 M glycine-HCl pH 2.2, 1 mg/ml BSA, incubated at 25° C. for10 min, and neutralized with 15 μL of 1M Tris-HCl pH 9.1. The selectedphage were amplified and titered. After four rounds of selection, thephage were cloned, individually amplified and sequenced.

(iv) Selection on Streptavidin Coated Magnetic Beads.

Magnetic beads Dynabeads M-280 streptavidin from Dynal Biotech wereused. The magnetic beads (50 μL) were washed twice with 200 μL of asolution containing 0.1 M NaOH, and 0.05 M NaCl, then the beads werewashed once with 200 μL of 0.1 M NaCl. Five washes with 200 μL of 1×binding buffer were performed. The beads were incubated for 3 min withshaking between washes. A counter selection was made againststreptavidin and plastic, by incubating the washed magnetic beads with2×10¹¹ phage at 25° C. for 30 min. The unbound phage were transferred toa tube containing 80 mM HEPES-KOH pH 7.4, 150 mM KCl, 20 mM MgCl₂, 100nM H18 RNA, 100 μg tRNA, and 40 U RNase Inhibitor. The mixture wasincubated at 25° C. for 30 mM, and in order to separate H18 RNA-boundphage and unbound phage the mixture was incubated with 50 μL of washedmagnetic beads at 25° C. for 30 min. Biotin at a concentration of 0.1 mMwas added and the beads were incubated at 25° C. for 5 min. The unboundphage were removed, and the beads were washed five times with 200 μL of1× wash buffer for beads (80 mM HEPES-KOH pH 7.4, 150 mM KCl, 20 mMMgCl₂, 0.1% tween-20 [v/v]), and three times with 200 μL of 1× bindingbuffer. The H18 RNA-bound phage were eluted with 100 μL of 0.2 Mglycine-HCl pH 2.2, 1 mg/ml BSA, at 25° C. for 10 min, and neutralizedwith 15 μL of 1 M Tris-HCl pH 9.1. The selected phage were amplified andtitered. Four rounds of selection were performed, increasing stringencyof washes for rounds two trough four by raising the concentration oftween-20 (v/v) in the wash buffer from 0.1% to 0.5%. The phage werecloned, individually amplified and sequenced. A fifth round of selectionwas performed, eluting the H18 RNA-bound phage by enzymatic degradationof the poly-dA tail with 250 U of DNase I from Fermentas Inc., at 37° C.for 30 min, then 1 μL of 0.5 M EDTA pH 8.0 was added. The phage werecloned, amplified and sequenced.

A separate panning was performed doing three rounds of selection, andeluting the bound phage using DNase I. Counter-selected phage wereincubated with 80 mM HEPES-KOH pH 7.4, 150 mM KCl, 20 mM MgCl₂, 250 nMH18 RNA, 100 μg tRNA, and 40 U RNase Inhibitor, at 25° C. for 30 min.The mixture was added to washed beads and incubated at 25° C. for 30min. Biotin at a concentration of 0.1 mM was added and incubated at 25°C. for 5 min. The beads were washed five times with 200 μL of 1× washbuffer for beads with 0.05% tween-20 (v/v), three times with 200 μL of1× binding buffer, once with 300 μL of 1× reaction buffer for DNase Ifrom Fermentas Inc., and the H18 RNA-bound phage eluted with 250 U ofDNase I at 37° C. for 30 min, then 1 μL of 0.5 M EDTA pH 8.0 was added.The phage were cloned, amplified and sequenced after rounds two andthree.

(v) Peptide Synthesis.

Select peptides were chemically synthesized by SynPep Corporation, andSigma-Genosys with amidated C-termini and acetylated N-termini.

(vi) E. coli Transcription/Translation Assay.

E. coli S30 extract system for Circular DNA was used from Promega.Experiments were carried out in 96-well conical 0.2 mL plates from DOTScientific Inc., in a final volume of 10 μL. S30 extract (3 μL) waspreincubated in the 96-well plates with 1 μL of water or peptides in arange of concentrations at 25° C. for 5 min. Translation mix (6 μL)containing 0.7 pig of pBESTluc™ DNA, 1 mM amino acid mixture withoutmethionine (0.8 μL), S30 premix (4.0 μL), and 2.5 pmol [³⁵S]methionine(1175 Ci/mmol) from MB Biomedicals was added to each well and incubatedat 37° C. for 5 min, then placed on ice. A solution of 1 M NaOH wasadded to the wells (245 μL) and incubated at 37° C. for 10 min.Synthesized protein was precipitated by addition of 1 ml of ice-cold 25%trichloroacetic acid (TCA)/2% casamino acids, and incubation on ice for30 min. The samples were filtered on glass fiber filters pre-wetted with5% TCA. The filters were washed three times with 5% TCA and once withacetone, dried and subjected to scintillation counting.

(vii) Eukaryotic Translation Assay.

The Promega flexi-rabbit reticulocyte lysate system was used.Experiments were carried out in 96-well conical 0.2 mL plates from DOTScientific Inc., in a final volume of 10 μl. Rabbit Reticulocyte Lysate(7 μL) was incubated with 1 μL of water or peptides in a range ofconcentrations at 25° C. for 5 min. Luciferase Control RNA (Promega) wasdenatured before use by incubating the RNA at 65° C. for 3 min, andimmediately placed on ice. Translation mix (2 μL) containing 0.3 μg ofdenatured Luciferase Control RNA, 1 mM amino acid mixture withoutmethionine (0.3 μL), 6.7 pmol [³⁵S]methionine (1175 Ci/mmol) from MBBiomedicals, 8 U Ribonuclease Inhibitor (Roche), and 2.5 M potassiumchloride (0.4 μL) was added to each well and incubated at 30° C. for 20min. The translation reaction was stopped by adding 10 μL ofcyclohexamide solution (100 μg/ml) and placing the wells on ice. Thetranslation assays were transferred to tubes containing 245 μL of 1 MNaOH and incubated at 37° C. for 10 min. Synthesized protein wasprecipitated by addition of 1 ml of ice-cold 25% trichloroacetic acid(TCA)/2% casamino acids, and incubation on ice for 30 min. The sampleswere filtered on glass fiber filters pre-wetted with 5% TCA. The filterswere washed three times with 5% TCA and once with acetone, dried andsubjected to scintillation counting.

(viii) Synergy Activity Testing of Peptides with Antibiotics.

Stock solutions of peptides and antibiotics were prepared and serialtwofold dilutions were made containing twice the desired finalconcentration. Drug combinations were prepared in 96-well conical 0.2 mlplates, and 1 μL of each combination was tested in the E. colitranscription/translation assay as described.

(ix) Determination of Peptide Dissociation Constants by FluorescenceSpectroscopy Using 30S Ribosomal Subunits and Peptides FluorescentlyLabeled.

30S subunits were obtained from E. coli MRE600, and purified by 10-40%(w/v) sucrose gradient. Peptides were chemically synthesized bySigma-Genosys, with amidated C-termini and a dansyl group at theN-termini. Fluorescent peptide solution for titration was prepared at aconcentration of 1 μM in TNM buffer (50 mM Tris-HCl pH 7.6, 100 mMNH4Cl, 10 mM MgCl₂), and 6 mM β-mercaptoethanol. The 30S subunits wereprepared in TNM buffer, placed at 42° C. for 2 min, then placed at roomtemperature. Aliquots of 30S subunits were added sequentially to thepeptide at room temperature. A Varian Cary Eclipse FluorescenceSpectrophotometer was used with an excitation wavelength of 325 nm andemission maxima at 560 nm.

Example 2 Peptide Selection Method

The following example shows one way the selection method of the presentinvention can be used. In this example, a novel target site in aribosomal unit was used to screen for peptides with affinity to thenovel target site. In this example, the extended target molecule is anRNA/DNA hybrid construct wherein the DNA is tagged with biotin. Variousother target molecules and affinity tags are contemplated in thisinvention as discussed above.

The affinity selection methodology was designed and developed using thetwo capture formats of magnetic beads and microtiter plates, and twoelution systems including nonspecific elution, and specific elution byenzymatic digestion of the specifically designed poly-dA tail of H18 RNAwith DNase I. H18 RNA at a concentration of 100 nM was allowed tointeract with the phage library in solution in the presence of tRNA (2μg/μL). The RNA-binding phage were captured using either capture format,and biotin was added to a final concentration of 0.1 mM in order todisplace any streptavidin-binding phage from the solid support. Unboundphage were washed from the solid support with wash buffer supplementedwith tween-20. The stringency of washes was increased for the magneticbeads capture system, by increasing the concentration of tween-20 from0.1% to 0.5% after the first round of selection. The stringency ofwashes for the microtiter well plate system was kept constant throughoutthe rounds of selection by using a concentration of tween-20 of 0.05%.The bound phage were released from the solid support using an acidicbuffer as a nonspecific eluant. After four rounds of selection thesequences of the phage-displayed peptides were obtained by sequencing ofthe phage DNA.

An additional round of selection was performed using the amplifiedeluate from the fourth round of magnetic beads selection. The boundphage were eluted by using DNase I. A separate affinity selection wascarried out using the magnetic beads capture system and eluting thebound phage specifically with DNase I. Three rounds of selection weredone. An overview of the selection process is shown in FIGS. 2A and B.

Twenty-six peptide motifs were obtained through the affinity selectionprocess (Tables 1 and 2). After four rounds of selection using magneticbeads and nonspecific elution, 15 peptide sequences were obtained (Table1), and the peptide motif AGAAMSH (BLS 15) (SEQ ID NO. 2) was the mostpredominant. Three different peptide sequences were obtained from thefourth round of well plate capture selection (Table 1), and the peptideAMSAPIP (BLS18) (SEQ ID NO. 3) was the most predominant sequence.Interestingly, BLS 15 and BLS 18 obtained from magnetic beads and wellplate selection respectively, contain a conserved three amino acidresidue motif (AMS), which upon detailed structural analysis will giveimportant information about the rules that govern peptide-RNArecognition, and will help in the development of new antibioticstargeting the new unexploited site H18 RNA.

Results from the magnetic beads selection with specific elution areshown in Table 2. After two rounds of selection seven peptide sequenceswere obtained, and the peptide sequence SILPYPY (SEQ ID NO. 4) (BLS26)was found to be the most predominant.

Alignment of the peptide sequences revealed the presence of severalconserved motifs among the peptides. Some common motifs observed wereAMS, HPP, THP, and LHL. Independently of the method of selection(magnetic beads or well plates), and elution (nonspecific or specific)conserved motifs were observed, such as peptides BLS18 (AMSAPIP) (SEQ IDNO. 3) and BLS2 (VQSLPSP) (SEQ ID NO.5) which were selected by wellplates and magnetic beads respectively and using nonspecific elution,sharing the common motif SXPXP (SEQ ID NO. 6) (X=any residue). Anothermotif selected was SXXLPT (SEQ ID NO. 7) from peptides BLS8 (LSPKLPT)(SEQ ID NO. 8) and BLS23 (SSLLPTT) (SEQ ID NO. 9), selected bynonspecific elution and specific elution respectively, using magneticbeads. Common motifs can be used in combinatorial screening assays tofind key residues within the motifs that are important for binding RNA,and this peptide scaffolds can also be used to build new drugs.

Peptide sequences obtained by the newly developed affinity selectionmethodology were synthesized to explore their ability as ribosomalinhibitors of protein synthesis, as well as to conduct more detailedstudies of the interaction of the selected peptides with H18 RNA.

Example 3 The Identified Peptides can Inhibit Protein Synthesis

The synthesized peptides were tested for their inhibitory activity inprotein synthesis using cell-free translation assays from E. coli. Thesystem uses a circular DNA template in a coupled in vitrotranscription/translation reaction for the production of the proteinluciferase. The system was supplemented with the radiolabeled amino acid[³⁵S]methionine to measure total production of luciferase in the assay.Inhibition of luciferase synthesis by the selected peptides was testedat a peptide concentration of 100 μM. As was expected from the importantfunctional role of H18-stem loop in the ribosomal subunit, inhibition ofprotein synthesis was observed. The strongest inhibitors of proteinsynthesis were peptides BLS15, BLS17, and BLS18 (FIG. 3) with a percentinhibition of 70-80%. Peptides BLS6, BLS14, and BLS20 inhibited proteinsynthesis by 30-40%. A 20% stimulation of protein synthesis was alsoobserved by peptides BLS25 and BLS26. Strikingly, peptides BLS15 andBLS18 show high potency for inhibition of protein synthesis with andIC₅₀ of 15 μM and 137 μM (FIG. 4), respectively, and were the mostcommon peptide found by magnetic bead and microtiter well captureselection. In addition, BLS 15 was shown to inhibit synthesis of GFP inthe coupled transcription-translation system. In accordance with thehigh conservation of H18 across the evolutionary domains, both peptidesinhibited protein synthesis in the rabbit reticulocyte cell-freetranslation system with IC₅₀ of 20 μM (BLS15) and 6.3 μM (BLS18) (FIG.5). Peptide BLS18 is a more potent inhibitor of protein synthesis in aneukaryotic system by 20-fold compared to a bacterial system, exhibitingits importance as a potential anticancer agent.

In an attempt to confirm the site of peptide binding, RNA footprintingwas performed. No changes in RNA reactivity was observed in the presenceof BLS15 or BLS18 with DMS, kethoxal, DEPC, or lead cleavage. Smallchanges in accessibility of 2′-hydroxyl of C522 and A523 toN-methylisatoic anhydride were observed in the presence of BLS15 as wellas in the presence of streptomycin.

Example 4 Synergism Between Identified Peptides and Antibiotics

Combination drug therapy for treating infectious diseases can be used tocounteract drug resistance. The activity of drugs in combination iscrucial for treating bacteria that show resistance to any singleantibiotic in clinical use, and is the last resource for treatingpatients until new antibiotics are discovered. The importance ofcombination therapies prompted us to test the selected peptides incombination with several antibiotics to investigate synergy ininhibition of protein synthesis in a bacterial system.

BLS15 was tested in combination with streptomycin, an antibiotic thatmakes contacts with 1-118 and interferes with decoding, and showedsynergistic action (FIG. 6). The potency of BLS15 in inhibiting proteinsynthesis in a bacterial system increased by 25-fold in the presence of3 μM streptomycin. Other related antibiotics were tested, and asynergistic response was also observed. The newly identified peptideshave a potential of being used in combination with other antibacterialsto fight resistant bacteria, and they can be used as well by linkingthem to other antibiotics to enhance potency creating a new class ofdrugs; this technique will enhance the affinity and specificity ofpeptide-RNA ligands. A similar example to our technique is the linkageof two ribosomal antibiotics, sparsomycin and linezolid, creating a newcompound that is active against a great variety of pathogens.

Example 5 Use of the Identified Peptides in Screening Assays

A method to study the interactions of the identified peptides with H18RNA was developed by tagging a fluorescent group to the peptide motif.Dansyl is a very sensitive fluorophore used to detect changes in themicroenvironment of proteins, and was used as a reporter group to detectchanges in the environment of identified peptides upon binding to thestem-loop H18 of 30S subunits. The advantage of this method is thatmodified peptides with a fluorophore are easy to synthesize, and it is adirect method of studying binding of peptides to ribosomes. The assay asdefined by the present invention can be carried out with differentdetection methodology depending on the detectable tag or label whichpreferably may be selected from the list including, but not limited to,fluorescent labels, chemiluminescent labels, colorimetric labels,enzymatic markers, and radioactive isotopes. For example, thefluorescent label can be selected from the list consisting of dansyl,fluorescein, Oregon green, rhodamine, tetra-methyl rhodamine, Texas-red,phycoerythrin, BODIPY fluorophore, and Eu³⁺.

The identified peptides can be used for screening of libraries ofsynthetic and natural organic compounds for drug leads affectingtranslation in bacterial and eukaryotic systems due to binding to H18and impairment of its activity in protein synthesis. The peptides can beused in ligand-displacement assays, or as surrogate ligands for highthroughput screening of small-molecule libraries. Detailed structuralinformation can be obtained from peptide-H18 complexes, such asthermodynamic and kinetic characterization, and will help inunderstanding ligand-RNA recognition and in the development of new classof RNA binders.

While the present invention has been described in terms of specificmethods and compositions, it is understood that variations andmodifications will occur to those skilled in the art upon considerationof the present invention. Those skilled in the art will appreciate, orbe able to ascertain using no more than routine experimentation, furtherfeatures and advantages of the invention based on the above-describedembodiments. Accordingly, the invention is not to be limited by what hasbeen particularly shown and described. All publications and referencesare herein expressly incorporated by reference in their entirety.

TABLE 1 Phage Peptide Sequence Found clone (SEQ ID NO:) (%)A. Magnetic Beads Capture Fourth Round (Nonspecific Elution) BLS1FPGHSGP (10) 2 BLS2 VQSLPSP (5) 2 BLS3 EPLQLKM (11) 2 BLS4 TPHNTST (12)2 BLS5 QWTWTQY (13) 2 BLS6 LTHPRWP (14) 2 BLS7 TKTDTWL (15) 2 BLS8LSPKLPT (8) 2 BLS9 NTPQGMT (16) 3 BLSlO THPLLLS (17) 7 BLS11GHWEARE (18) 7 BLS12 AVPRASF (19) 7 BLS13 YHPMPVP (20) 8 BLS14TPTTDGP (21) 20 BLS15 AGAAMSH (2) 32 Fifth Round (DNase I Elution) BLS19VHRHPPH (22) 4 BLSlO THPLLLS (23) 7 BLS11 GHWEARE (24) 11 BLS15AGAAMSH (2) 21 BLS7 TKTDTWL (25) 29 BLS14 TPTTDGP (26) 29B. Microtiter Plates Capture Fourth Round (Nonspecific Elution) BLS16MKHPPRI (27) 2 BLS17 GTMLAAV (28) 4 BLS18 AMSAPIP (3) 94

TABLE 2 Phage Peptide Sequence Found clone (SEQ ID NO:) (%)Second Round (DNase I Elution) BLS20 TMTPPTR (29) 6.6 BLS21 GNDWPHW (30)6.6 BLS22 EHPYITV (31) 6.6 BLS23 SSLLPTT (9) 6.6 BLS24 NTNTLHL (32) 6.6BLS25 SYPDLHL (33) 6.6 BLS26 SILPYPY (4) 60Third Round (DNase I Elution) BLS26 SILPYPY (4) 100

1. An isolated therapeutic peptide composition comprising: a sequence ofat least seven amino acids capable of inhibiting protein synthesisthrough an interaction at a stem-loop H18 in 16S rRNA of a 30S ribosomalsubunit.
 2. The therapeutic peptide composition of claim 1, wherein thepeptide exhibits at least about 25 percent inhibition of proteinsynthesis in a cell-free translational assay.
 3. The therapeutic peptidecomposition of claim 1, wherein the peptide exhibits at least about 50percent inhibition of protein synthesis in a cell-free translationalassay.
 4. The therapeutic peptide composition of claim 1, wherein thepeptide is in the range of about seven to about thirty amino acidresidues and selectively binds to the stem-loop H18 in 16S rRNA of the30S ribosomal subunit.
 5. The therapeutic peptide composition of claim1, wherein the peptide sequence comprises at least one sequence selectedfrom the group consisting of SEQ ID NOS: 2-33.
 6. The therapeuticpeptide composition of claim 1, wherein the peptide sequence comprisesat least one of the amino acid motifs selected from the group consistingof AMS, HPP, THP, LHL, SXPXP (SEQ ID NO: 6) and SXXLPT (SEQ ID NO: 7).7. The therapeutic peptide composition of claim 1, wherein the peptidesequence comprises at least one sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO:
 28. 8. Thetherapeutic peptide composition of claim 1, further comprising apharmaceutically acceptable carrier.
 9. The therapeutic peptidecomposition of claim 1, wherein the peptide is coupled to a moietycapable of directing the peptide to a target cell. 10-24. (canceled) 25.A method for treating a bacterial infection in a subject comprisingadministrating the therapeutic peptide composition of claim
 1. 26.(canceled)
 27. (canceled)
 28. The method of claim 25, wherein thepeptide is delivered in an amount sufficient to inhibit the growth ofbacteria in vivo.
 29. The method of claim 25, wherein said peptide isdelivered locally or regionally to a site of infection.
 30. The methodof claim 25, wherein said peptide is administered to a wound site. 31.The method of claim 25, wherein said peptide is administered topically.32. The method of claim 25, wherein said peptide is deliveredsystemically.
 33. The method of claim 25, wherein said peptide isdelivered via intravenous or intra-arterial injection.
 34. The method ofclaim 25, further comprising administering to said subject one or moreantimicrobial compounds.
 35. A method for preventing a microbialinfection in a subject comprising administrating the therapeutic peptidecomposition of claim 1 in an amount sufficient to inhibit the growth ofmicrobes in vivo.
 36. A method for preventing microbial growth in asolution comprising mixing said solution with the therapeutic peptidecomposition of claim 1 in an amount sufficient to inhibit the microbialgrowth in said solution.
 37. A method for preventing bacterialattachment or growth on an abiotic surface comprising coating saidsurface with the therapeutic peptide composition of claim 1 in an amountsufficient to inhibit the growth of bacteria on said abiotic surface.38. The method of claim 37, wherein said surface is part of a medicaldevice.
 39. The method of claim 37, wherein said medical device is asyringe, a stent, a catheter, fluid container, a pacemaker, or animplantable pump. 40.-48. (canceled)